Heparin-induced thrombocytopenia and thrombosis (HIT/T), a serious complication of heparin therapy, is one of the most common antibody-mediated thrombocytopenias and can result in life and limb- threatening thrombosis. We have generated and characterized the first mouse model to recapitulate the salient features of the disease and confirmed that complexes of heparin and platelet factor 4, antibodies to the complex, and Fc-gammaRIla-dependent platelet activation are both necessary and sufficient to model the disease in vivo. However, many questions of paramount importance remain, including: Why do only some patients with HIT antibodies become thrombocytopenic? And, why does a subset of the thrombocytopenic patients develop the more serious complication of thrombosis? To address these questions we propose to use our mouse model to systematically investigate the contributions of specific features of heparin/PF4 antibodies and the role of an intact, yet activated endothelium in the pathogenesis of HIT/T. In Specific Aim 1, we will determine the extent to which intrinsic characteristics of human heparin/PF4 antibodies contribute to thrombosis. We have developed and used our novel hybridoma method to clone native human heparin/PF4 antibodies from patients with HIT/T. We have recently cloned a human heparin/PF4 IgG antibody that binds PF4 in a heparin-dependent fashion. We will use this method to clone additional authentic human heparin/PF4 antibodies in quantities sufficient for systematic characterization of their IgG subclass, variable chain gene usage, PF4 binding epitope, and binding affinity. We will also examine their ability to activate platelets and endothelial cells in vitro. These studies will form the basis for determining the antibody properties that contribute to thrombosis in vivo as proposed in Specific Aim 2. In Specific Aim 2, we will study the association of specific properties of our cloned human heparin/PF4 antibodies and a pro- thrombotic milieu with the incidence and severity of thrombosis in vivo. Pro-thrombotic conditions will be induced by dietary and genetic models of atherosclerosis to determine the influence of an activated endothelium in the pathogenesis of HIT/T in vivo. These results of our studies of heparin/PF4 antibodies and their interaction with the vascular endothelium will provide insight into the thrombosis in patients with HIT/T. This new information may facilitate development of improved diagnostic tests and therapy for patients at risk for heparin-induced life-threatening thrombosis.